This proposal concerns the development of an accurate voltage clamp of cardiac cell type: the isolated canine cardiac Purkinje cell, and the use of this system to measure the cells. Purkinje cells will be enzymatically isolated and kept under tissue culture conditions. Electron microscopy will be used to study the morphology of the cells and any alterations that follow isolation. Electrical properties of isolated cells will be compared to those of the "standard", artificially shortened, Purkinje fiber. The cells will be voltage clamped with two micro electrodes. Measurements of the small signal impedance will be used to test the frequency response of the clamp-cell system in order to establish standards of accuracy. A variety of standard voltage clamp experiments will be done to measure the kinetics of each channel system using ionic substitution and pharmacologic agents to isolate currents to a single ionic species. An attempt will be made to develop an internally dialyzed cardiac cell preparation using the system developed by Kostyuk et al. (1975) for molluscan neurones. If successful, the dialyzed preparation will be used to investigate the effects of intracellular calcium levels on potassium conductance and the effects of intracellular cyclic nucleotides. Current fluctuation analysis will be used to characterize the channel conductances and density, in particular to define whether all the observed potassium currents pass through the same channels.